Independent validation of Deverman delivery platform using engineered AAVs to deliver CRSIPR/Cas9 to mouse brain | SCGE Toolkit

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Validation Experiment: Independent validation of Deverman delivery platform using engineered AAVs to deliver CRSIPR/Cas9 to mouse brain

PI: Jason D Heaney, PhD

Description: Validation of delivery of AAV custom designed to cross the blood-brain barrier for CRISPR/Cas9 editing. Editing detected and quantified in brain by generation of tdTomato fluorescent protein signal from Ai9 reporter mice

Delivery Assays: Identification of of GFP-positive nuclei (green)

Editing Assay:

Qualitative assesment of tdTomato-negative cells throughout the entire brain
Quantitative assessment of tdTomato-positive cells per DAPI-stained nucleus in a specific brain region
Qualitative assesment of prevalence of tdTomato-positvie cells throughout the entire brain

Parent Project: BCM-Rice Resource for the Analysis of Somatic Gene Editing in Mice - Deverman Validation (Deverman, Benjamin)

Download: Submitted files

Original Experiment/s that are being validated:

Testing gRNA sequence and gRNA scaffold modified in Ai9 mice.

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Results

Independent_validation_of_Deverman_delivery_platform_using_engineered_AAVs_to_deliver_CRSIPR/Cas9_to_mouse_brain
											Editing Efficiency		Delivery Efficiency
Record Id	Condition	Tissue	Cell Type	Sex	Editor	Model	Target Locus	Guide	Vector	Dosage	Injection Frequency	Editing Efficiency Signal	Editing Efficiency % Area	Delivery Efficiency Signal	Image
15000001448	Arm1a: BI28 Neuron Specific Promoter, Experimental Animals, Males	brain (TARGET)	neuron	Male	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once	Present; Present; Present; Present; Present;
15000001449	Arm1a: BI28 Neuron Specific Promoter, Experimental Animals, Males	brain (TARGET)	neuron	Male	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once			Present; Present; Present; Present; Present;
15000001450	Arm1a: BI28 Neuron Specific Promoter, Experimental Animals, Females	brain (TARGET)	neuron	Female	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once	Present; Present; Present; Present; Present;
15000001451	Arm1a: BI28 Neuron Specific Promoter, Experimental Animals, Females	brain (TARGET)	neuron	Female	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once			Present; Present; present; present; present;
15000001452	Arm 1b: BI28 Neuron Specific Promoter, Positive Delivery Control Animals, Males	brain (TARGET)	neuron	Male	SaCas9	Ai9 mouse			BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2	0.25E+11 viral genomes/gram for each virus	once			present; present; present; present;
15000001453	Arm 1b: BI28 Neuron Specific Promoter, Positive Delivery Control Animals, Females	brain (TARGET)	neuron	Female	SaCas9	Ai9 mouse			BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2	0.25E+11 viral genomes/gram for each virus	once			present; present;
15000001454	Arm 2a: BI28 Astrocyte Specific Promoter, Experimental Animals, Males	cerebral cortex (TARGET)	astrocyte	Male	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once		35.0; 45.9; 33.3; 41.8; 29.7; 46.3;
15000001455	Arm 2a: BI28 Astrocyte Specific Promoter, Experimental Animals, Males	dorsal plus ventral thalamus (TARGET)	astrocyte	Male	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once		53.5; 29.5; 29.2; 53.8; 46.4; 48.0;
15000001456	Arm 2a: BI28 Astrocyte Specific Promoter, Experimental Animals, Males	brain (TARGET)	astrocyte	Male	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once			Present; Present; Present; Present; Present; Present;
15000001457	Arm 2a: BI28 Astrocyte Specific Promoter, Experimental Animals, Females	cerebral cortex (TARGET)	astrocyte	Female	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once		43.1; 37.3; 39.5; 41.4; 32.8; 36.8;
15000001458	Arm 2a: BI28 Astrocyte Specific Promoter, Experimental Animals, Females	dorsal plus ventral thalamus (TARGET)	astrocyte	Female	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once		43.7; 52.2; 56.1; 51.1; 48.5; 51.6;
15000001459	Arm 2a: BI28 Astrocyte Specific Promoter, Experimental Animals, Females	brain (TARGET)	astrocyte	Female	SaCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	L1-modified; L2-modified	BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2 ; BI28:AAV-GFAP-SaCas9-WPRE3-pA	0.25E+11 viral genomes/gram for each virus	once			present; present; present; present; present; present;
15000001460	Arm 2b: BI28 Astrocyte Specific Promoter, Positive Delivery Control Animals, Males	brain (TARGET)	astrocyte	Male	SaCas9	Ai9 mouse			BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2	0.25E+11 viral genomes/gram for each virus	once			present; Present; Present;
15000001461	Arm 2b: BI28 Astrocyte Specific Promoter, Positive Delivery Control Animals, Females	brain (TARGET)	astrocyte	Female	SaCas9	Ai9 mouse			BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2	0.25E+11 viral genomes/gram for each virus	once			Present; Present; Present;
15000001462	Arm 3: Negative Control Saline, Males	brain (TARGET)		Male		Ai9 mouse					once	absent; absent; absent;
15000001463	Arm 3: Negative Control Saline, Males	brain (TARGET)		Male		Ai9 mouse					once			absent; absent; absent;

Title	Description	File Download	SCGE ID
Heaney_SATC Tissue Processing, Imaging and Analysis	Procedure for tissue preparation, imaging and analysis.	Heaney_SATC_Tissue_Processing_Imaging_and_Analysis.pdf	21000000025